Aggregation and deposition of misfolded proteins, known as amyloid, is a pathophysiological hallmark of approximately 20 human disorders collectively known as amyloid diseases (1-3). Human Islet Amyloid Poly Peptide (hIAPP) is a natively unstructured pancreatic endocrine hormone that regulates food intake and fat storage. It is co-secreted with insulin from pancreatic β-cells in response to glucose intake. In Type 2 diabetes mellitus (T2DM), hIAPP forms amyloid fibrils in the islets of Langerhans. Under the conditions of T2DM, hIAPP amyloid deposits in the Islets are toxic to β-cells. Several human and animal studies show that amyloid forms before β-cell deficiency and that the formation of oligomers and intracellular aggregates induces β-cell death (4, 5). In both type 1 (T1DM) and T2DM, IAPP release diminishes in parallel with the decrease in insulin release and conversely, IAPP levels are elevated in conditions of insulin resistance (high insulin secretion).
Most oral diabetes drugs lower blood glucose by stimulating the pancreas to release more insulin. Unfortunately, increased release of insulin is accompanied by increased release of hIAPP, which results in enhanced amyloid deposition, in turn leading to β-cell death and loss of natural insulin production. Therefore, these drugs do not cure the underlying cause of reduced insulin secretion. Rather, they manage hyperglycemia initially by stimulating insulin release, but in effect worsen the underlying problem and become progressively ineffective as β-cells die. This gradual loss of β-cells results in conversion of T2DM patients to iatrogenic T1DM patients (T2-T1DM) who require exogenous insulin supplements for blood glucose maintenance. In fact, drugs like Rosiglitazone and Metformin that work by improving insulin sensitivity and thereby reduce insulin demand have been shown to reduce the prevalence and severity of islet amyloid in transgenic mice. Several animal and human studies show that amyloid forms before β-cell deficiency and that formation of intracellular aggregates induces β-cell death. Amyloid fibrils and their pre-fibrillar aggregates exhibit toxicity in cell culture-based assays and it is thought that prevention of aggregation of pathogenic amyloid peptides might prevent disease progression.
Calnuc or Nucleobindin 1 (NUCB1) is a 55 kDa protein, which was first reported to be a growth and differentiation factor associated with lupus syndrome (Kanai et al., Immunol. Lett., 32, 43-48, 1992). Calnuc acquires its name from its DNA-binding and calcium binding ability. The NUCB1 (i.e. Calnuc) domain structure comprises, from N-terminus to C-terminus, a signal sequence at its N-terminus followed by a DNA binding domain of basic residues, an N-terminal proximal EF hand domain comprising a helix-loop-helix motif, an intervening acidic region, a second C-terminal proximal EF hand domain comprising a helix-loop-helix motif, and a leucine zipper domain (Miura et al., Biochem. Biophys. Res. Commun., 187, 375-380, 1992). Both the DNA binding domain and leucine zipper are crucial for binding of NUCB1 to DNA.
Recent studies have suggested that overexpression of NUCB1 down-regulates the mRNA production of Amyloid precursor protein (APP) and inhibits its biosynthesis (Lin et al., J. Neurochem., 100, 1505-14, 2007). Aggregation of Aβ isoforms generated from the sequential proteolytic cleavage of APP by β-(Beta Amyloid Cleaving Enzyme-1) and γ-secretase has been well characterized in Alzheimer's disease. In addition, abnormal calcium homeostasis has also been observed in the brains of demented patients. One study indicated that: i) NUCB1 binds to APP in a calcium dependent manner where binding is inhibited by Ca+2; ii) NUCB1 co-localizes with APP in vivo; iii) NUCB1 regulates APP protein levels by affecting APP synthesis; and that iv) the expression level of NUCB1 is decreased in the brains of Alzheimer's disease patients by 50% (Lin et al., J. Neurochem., 100, 1505-14, 2007). Nonetheless, Lin et al., (J. Neurochem., 100, 1505-14, 2007) did not indicate that NUCB1 could disaggregate amyloid fibrils or inhibit amyloid fibril formation.
It has also been shown that both: (i) Ca2+-free soluble NUCB1 (sNUCB1) comprising an EF hand loop 1 domain, an intervening acidic region, an EF hand loop 2 domain; and, (ii) engineered sNUCB1 (tetramutant) comprising mutations in the EF-hand loop domains can inhibit the aggregation of both hIAPP and Aβ42 and decrease their toxicity in cell viability assays (WO 2010/009330). Ca2+-free soluble sNUCB1 and the sNUCB1 (tetramutant) were also shown to disaggregate existing amyloid fibrils (WO 2010/009330). Various sNUCB1 mutants comprising an EF hand loop 1 domain, an intervening acidic region, an EF hand loop 2 domain but lacking sNUCB1 C-terminal residues 408-461 (sNUCB1(G408Ter), 401-461 (sNUCB1(Q401Ter)), and 333-461 (sNUCB1(W333Ter)) were also shown to inhibit aggregation of hIAPP (WO 2010/009330).
Using several amyloidogenic peptides, including hIAPP, engineered soluble forms of expressed Nucleobindin-1 (sNUCB1), a ubiquitous Ca2+-binding protein, can inhibit amyloid formation and even disaggregate preformed fibrils (10). The mechanism of action of sNUCB1 is novel. The sNUCB1 protein apparently “caps” toxic protofibrillar species, reduces their toxicity, and prevents them from growing into mature fibrils. The sNUCB1-“capped” protofibrils are stable and can be isolated. The sNUCB1 binds to protofibrils that originate from amyloidogenic peptides, but it does not bind to the peptides themselves (10).
Conformation-specific antibodies have proven to be a powerful tool for detecting generic epitopes common to fibrils and are potentially therapeutic agents (6, 7, US Pat. Appl. Pub. No. 20100209422). Together, these antibodies have been useful in basic research to detect specific structural states of amyloidogenic proteins, and to shed light on the pathway of protein aggregation that leads to amyloid fibril formation. For example, the A11 antibody has been reported to specifically recognize a generic epitope common to prefibrillar oligomers of Aβ42 and not fibrils, monomers or natively folded Alzheimer's precursor proteins (8). In Alzheimer's Disease brain tissue, A11 stains punctate deposits but not diffuse plaques, indicating that diffuse deposits do not contain prefibrillar oligomers. These studies show the power of conformation-specific antibodies in teasing apart structural subtleties related to the molecular pathophysiology of amyloid disease.